1. Field of the Invention
The present invention relates generally to the fields of cancer biology and drug delivery and, more specifically, to peptides that selectively home to a tumor, particularly a malignant tumor, to compositions comprising an agent such as a therapeutic agent conjugated to such tumor homing molecules, and to methods of using such molecules to target an agent to a tumor.
2. Background Information
Continuous developments over the past quarter century have resulted in substantial improvements in the ability of a physician to diagnose a cancer in a patient. For example, antibody based assays such as that for prostate specific antigen now allow early diagnosis of cancers such as prostate cancer. More recently, methods of genetic screening are becoming available to identify persons that may be particularly susceptible to developing a cancer. Genetic screening methods are based on the identification of one or more mutations in a gene that correlates with the development of a cancer. For example, the identification of genes such as BRCA1 and BRCA2 allowed the further identification of mutations in these genes that, in some cases, can correlate with susceptibility to developing breast cancer.
Unfortunately, methods for treating cancer have not kept pace with those for diagnosing the disease. Thus, while the death rate from various cancers has decreased due to the ability of a physician to detect the disease at an earlier stage, the ability to treat patients presenting with more advanced disease has advanced only minimally.
A major hurdle to advances in treating cancer is the relative lack of agents that can selectively target the cancer, while sparing normal tissue. For example, radiation therapy and surgery, which generally are localized treatments, can cause substantial damage to normal tissue in the treatment field, resulting in scarring and, in severe cases, loss of function of the normal tissue. Chemotherapy, in comparison, which generally is administered systemically, can cause substantial damage to organs such as bone marrow, mucosae, skin and the small intestine, which undergo rapid cell turnover and continuous cell division. As a result, undesirable side effects such as nausea, loss of hair and drop in blood cell count occur as a result of systemically treating a cancer patient with chemotherapeutic agents. Such undesirable side effects often limit the amount of a treatment that can be administered. Thus, cancer remains a leading cause of patient morbidity and death.
Efforts have been made to increase the target specificity of various drugs. For example, where a unique cell surface marker is expressed by a population of cells making up a tumor, an antibody can be raised against the unique marker and a drug can be linked to the antibody. Upon administration of the drug/antibody complex to the patient, the binding of the antibody to the marker results in the delivery of a relatively high concentration of the drug to the tumor. Similar methods can be used where a particular cancer cell or the supporting cell or matrix expresses a unique cell surface receptor or a ligand for a particular receptor. In these cases, the drug can be linked to the specific ligand or to the receptor, respectively, thus providing a means to deliver a relatively high concentration of the drug to the tumor.
Tumors are characterized, in part, by a relatively high level of active angiogenesis, resulting in the continual formation of new blood vessels to support the growing tumor. Such angiogenic blood vessels are distinguishable from mature vasculature. One of the distinguishing features of angiogenic vasculature is that unique endothelial cell surface markers are expressed. Thus, the blood vessels in a tumor provide a potential target for directing a chemotherapeutic agent to the tumor, thereby reducing the likelihood that the agent will kill sensitive normal tissues. Furthermore, if agents that target the angiogenic blood vessels in a tumor can be identified, there is a likelihood that the agents can be useful against a variety of different types of tumors, since it is the target molecules in the angiogenic vessels that are recognized by such agents and not receptors specific for the tumor cells. However, the use of molecules that can bind specifically to tumor vasculature and target a chemotherapeutic agent to the tumor has not been demonstrated.
While linking a drug to a molecule that homes to a tumor can provide significant advantages for treatment over the use of a drug, alone, use of this method is severely limited by the scarcity of useful cell surface markers expressed in a tumor. Thus, a need exists to identify molecules that can selectively home to a tumor, particularly to the vasculature supporting the tumor. The present invention satisfies this need and provides related advantages as well.
The present invention provides a method of identifying a tumor homing molecule that homes to angiogenic vasculature of a tumor. The method includes the steps of contacting a substantially purified NGR receptor with one or more molecules and determining specific binding of a molecule to the NGR receptor, where the presence of specific binding identifies the molecule as a tumor homing molecule that homes to angiogenic vasculature of a tumor. In a method of the invention, the substantially purified NGR receptor can be, for example, CD13/aminopeptidase N. If desired, the substantially purified NGR receptor can be immobilized on a support such as a plate or a bead.
The invention also provides a method of identifying a homing molecule that homes to angiogenic vasculature using substantially purified NGR receptor. The method includes the steps of contacting a substantially purified NGR receptor with one or more molecules and determining specific binding of a molecule to the NGR receptor, where presence of specific binding identifies the molecule as a homing molecule that homes to angiogenic vasculature. The invention provides homing molecules that home to non-tumor angiogenic vasculature.
The present invention also provides a method of directing a moiety to angiogenic vasculature of a tumor in a subject by administering to the subject a conjugate including a moiety linked to a tumor homing molecule that exhibits specific binding to an NGR receptor, whereby the moiety is directed to angiogenic vasculature of a tumor. In a method of the invention, the tumor homing molecule can be, for example, a peptide containing the sequence NGR, and, if desired, can be part of a conjugate in which the moiety is a cytotoxic agent, drug or cancer therapeutic agent, for example, doxorubicin. A tumor homing peptide containing the sequence NGR can have, for example, the sequence CNGRCVSGCAGRC (SEQ ID NO:3), NGRAHA (SEQ ID NO:6), CVLNGRMEC (SEQ ID NO:7) or CNGRC (SEQ ID NO:8). In a method of the invention for directing a moiety to angiogenic vasculature of a tumor in a subject, the tumor homing molecule also can be, for example, an aminopeptidase inhibitor such as bestatin, o-phenanthroline, actinonin, amastatin, 2,2xe2x80x2-dipyridyl or fumagillin and can be linked, if desired, to a drug moiety.
Further provided herein is a method of imaging the angiogenic vasculature of a tumor in a subject by administering to the subject a conjugate having a detectable moiety linked to a tumor homing molecule that exhibits specific binding to an NGR receptor, whereby the conjugate selectively binds the angiogenic vasculature, and detecting the conjugate. A detectable moiety for imaging angiogenic vasculature can be, for example, a radionuclide. A useful tumor homing molecule for imaging angiogenic vasculature can be, for example, a peptide containing the sequence NGR, such as a peptide containing the sequence CNGRCVSGCAGRC (SEQ ID NO:3), NGRAHA (SEQ ID NO:6), CVLNGRMEC (SEQ ID NO:7) or CNGRC (SEQ ID NO:8). A tumor homing molecule for imaging angiogenic vasculature also can be, for example, an aminopeptidase inhibitor such as bestatin, o-phenanthroline, actinonin, amastatin, 2,2xe2x80x2-dipyridyl, fumagillin or another molecule that inhibits an aminopeptidase.
The invention also provides inhibitors of angiogenesis that are NGR receptor binding molecules. Such inhibitors can be, for example, an NGR receptor antibody or an aminopeptidase inhibitor such as bestatin, o-phenanthroline, actinonin, amastatin, 2,2xe2x80x2-dipyridyl or fumagillin, or a conjugate of such angiogenesis inhibitors to a drug or toxin.